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Subchronic olanzapine exposure leads to increased expression of myelination-related genes in rat fronto-medial cortex

Subchronic olanzapine exposure leads to increased expression of myelination-related genes in rat... Schizophrenia is a psychotic disorder with severe and disabling symptoms, such as hallucinations, delusions, blunted affect and social withdrawal. The neuropathology remains elusive, but disturbances in immunity-related processes, neuronal connectivity and myelination have consistently been linked to schizophrenia. Antipsychotic drugs can be efficient in reducing symptoms, acting primarily on the dopamine system, but additional biological targets are likely to exist. Here we have screened for novel mechanisms of action in an animal model, using adult rats exposed to long- acting olanzapine, achieving stable and clinically relevant antipsychotic drug concentrations. By microarray-based examination of global gene expression in the fronto-medial cortex, at the single gene- and gene-set level, we observed downregulation of two neuropeptide-encoding genes, Vgf and Cort (fold change −1,25 and −1,48, respectively) in response to olanzapine exposure. Furthermore, we demonstrated significant upregulation of five out of ~2000 GO predefined gene sets after olanzapine exposure. Strikingly, all were linked to myelination and oligodendrocyte development; “Ensheathment of neurons”, “Axon ensheathment”, “Myelination”, “Myelin sheath” and “Oligodendrocyte development” (FDR-values < 25). Sixteen of the leading edge genes in these gene sets were analysed independently by qPCR, of which 11 genes displayed significant upregulation, including Plp1, Mal, Mag and Cnp (fold change: 1,30, 1,50, 1,30 and 1,15, respectively). Several of the upregulated genes (e.g. MAG, MAL and CNP) have previously been reported as downregulated in post-mortem brain samples from schizophrenia patients. Although caution needs to be taken when extrapolating results from animal studies to humans, the data suggest a role for olanzapine in alleviating myelination-related dysfunction in schizophrenia. Introduction maturation, including myelination. Although the clinical Schizophrenia is a disabling psychiatric disorder with a manifestations have been known for centuries, the lifetime prevalence of about 0.7% . The illness is char- underlying pathophysiology and aetiology of the disorder acterised by complex clinical symptoms, including hallu- remain largely unknown. While the estimated heritability 2,3 cinations, delusions, social withdrawal and blunted affect, ranges from 60 to 80% , and recent studies have started 4–8 in addition to cognitive impairment. The onset of schi- to disclose genetic risk factors of schizophrenia , much zophrenia is normally during late adolescence or early still remains to be elucidated. adulthood, a time period coinciding with brain As an alternative approach to study the pathological disease mechanisms, large-scale gene expression studies have been performed on post-mortem brain samples from Correspondence: Kari M Ersland (kari.ersland@uib.no) patients suffering from schizophrenia. The most con- Dr. Einar Martens Research Group for Biological Psychiatry, Center for Medical sistent observations from these studies include alterations Genetics and Molecular Medicine, Haukeland University Hospital, Bergen 5021, in expression levels for genes related to brain white matter Norway The Norwegian Centre for Mental Disorders Research (NORMENT) and the K.G. and oligodendrocytes, signalling and synapses, GABA and Jebsen Centre for Psychosis Research, Department of Clinical Science, glutamate neurotransmission, mitochondrial function, in University of Bergen, Bergen, Norway © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Translational Psychiatry 1234567890 1234567890 Ersland et al. Translational Psychiatry (2017) 7:1262 Page 2 of 10 9–11 29,30 addition to immune and stress-response (reviewed in ). stimulating effects . We demonstrate a significant In particular, several studies have reported a down- downregulation of two neuropeptide-encoding genes, and regulation of genes linked to myelination and myelin- interestingly, a striking upregulation of gene sets linked to 12–16 producing oligodendrocytes in schizophrenia . Oligo- myelination-related functional pathways. To our knowl- dendrocyte membranes wrap around axons forming a edge, this is the first study to report on global gene multi-lamellar myelin structure, which is essential for expression alterations in rat brain in response to stable rapid propagation of action potentials and the long-term and clinically relevant olanzapine levels. integrity of neuronal axons. Since myelin is a lipid-rich membrane structure, it is interesting that several anti- Materials and methods psychotic drugs, especially clozapine and olanzapine, have Animals and drug exposure scheme been shown to have lipid-stimulating effects. The experiment was approved by, and carried out in The results from post-mortem brain studies have, accordance with the guidelines of the Norwegian Com- however, been challenging to replicate, probably attrib- mittee for Experiments on Animals (Forsøksdyrutvalget, uted to cofounding factors, e.g. age, severity of disease, FDU), following standardised application through the treatment with antipsychotic drugs, co-morbidity, animal facility at Haukeland University Hospital (ID post-mortem sample handling and type of microarray 2014–6735). Ten to 12 weeks old female outbred Sprague- platform used . Obstacles such as these are less promi- Dawley rats (weighing ~240 g) (Mollegaard, Denmark) nent when using animals to explore drug-induced were housed under standard conditions with an artificial changes in gene expression in the brain, although such 12:12 h light/dark cycle under constant 48% humidity. models have obvious limitations in the study of psychia- Female rats were selected as the administration of long- tric disorders . Rodents, primarily rats, have been acting olanzapine has resulted in clinically relevant extensively used to analyse the effect of various anti- metabolic phenotypes, while exposure of male rats have 26–28,31 psychotic drugs on gene expression in different areas of yielded less clear-cut results . Five animals were the brain. Overall, the alterations identified seem to housed in each cage, with free access to standard converge on some of the same pathways as observed in laboratory chow (Special Diets Services, Witham, UK) and patient post-mortem studies, including neurotransmis- tap water. Animals were randomly divided into two sion, neural plasticity, cell survival, ionic homoeostasis groups (n = 10) (the investigators were blinded to the 11,18–21 and synaptic functions . group allocations); the first group received a single Still, direct comparison between animal studies have intramuscular injection of commercially available olan- been problematic due to differences in methodology, zapine pamoate depot formulation (100 mg/kg BW brain areas examined, choice of antipsychotic drugs and ZypAdhera ) (Eli Lilly, Indianapolis, IN, USA), while the treatment schemes. The rapid metabolism of anti- second group received commercially available vehicle psychotic agents in rodents represents an additional solution (injection volume 160 μl/250 g BW) on the first challenge, impending clinically relevant serum con- day of the experimental period, as previously described . centrations of antipsychotic drugs, especially over time. Sample size and the injected dose were determined based 26,28 As an example, the half-life (t ) of the second-generation on previous experiments . Food was removed 10 h 1/2 antipsychotic olanzapine in rat serum is 3 h, while the before sacrifice, in order to reduce potential variability in 22–24 average t is 30 h in humans . Most global gene gene expression caused by differences in metabolic para- 1/2 expression studies to date have used daily oral adminis- meters. Based on previous rat experiments of gene tration of antipsychotic agents (mainly through drinking expression in peripheral tissues, a time period of 5 days 26,28 water, but also by gavage) or daily injections. Recently, our was selected, representing a sub-acute exposure time . group demonstrated that the use of depot injections of Care was taken to ensure minimal suffering of the animals atypical antipsychotics in rat leads to clinically relevant at all stages of the experiments. and stable serum concentrations over time, as compared to the fluctuating and often low serum drug concentra- Tissue dissection tions observed in rats exposed to twice-daily oral drug Rats were anesthetised by isoflurane gas (Isoba vet; 25–28 administration during the same time period . Schering-Plough, Denmark), and subsequently sacrificed In this work, we exposed rats to a 5-day treatment with by decapitation. Brains were removed from the skull, long-acting olanzapine to examine alterations in global washed in cold phosphate-buffered saline and placed on gene expression in the fronto-medial parts of the cerebral ice. Tissue samples from the fronto-medial part of the cortex, identifying drug-induced molecular profiles. We cortex (FMCx), striatum and hippocampus were dissected chose to study olanzapine, since this antipsychotic drug from the right hemisphere. In addition, the whole hypo- has been shown to be one of the most efficacious anti- thalamus was dissected. Tissue samples were immediately psychotic agents, in addition to displaying strong lipid- frozen on dry ice, and stored at −80 °C. Translational Psychiatry Ersland et al. Translational Psychiatry (2017) 7:1262 Page 3 of 10 Measurement of plasma olanzapine levels and hybridisation) and to obtain a normal distribution, Truncal blood was collected as previously described . inter-array quantile normalisation and log2 transforma- Plasma levels of olanzapine were determined by means of tion was performed, respectively. Identification of single high-performance liquid chromatography using Agilent microarray probes that differed significantly in expression 1290 Infinity Binary LC, coupled to an Agilent 6490A level (i.e. hybridisation signal intensity) between sample triple quadrupole mass spectrometer using positive elec- groups was carried out by significance analysis of micro- trospray ionisation (Agilent Technologies, Santa Clara, arrays (SAM) , comparing signal intensities of all probes CA, USA). across all samples. In order to reduce the number of false- positive findings, the significance threshold was set to a RNA extraction highly conservative false discovery rate (FDR) of 0 and Tissue samples from FMCx, hypothalamus, hippo- 1000 permutations were performed. Statistically sig- campus and striatum were homogenised using a Tissue- nificant gene expression profiles were further explored Lyser (Qiagen, Hilden, Germany). RNA extraction was and curated by manual inspection. Gene set enrichment performed on an ABI Prism™ 6100 Nucleic Acid Pre- analysis (GSEA) was used to analyse the global gene pStation (Applied Biosystems, Foster City, CA, USA), with expression data, at the gene-set level. A priori defined DNase treatment according to the manufacturer’s proto- gene sets were based on Gene Ontology (GO) annota- col. Amount and quality of total RNA was measured using tions, implemented in J-Express . In GO, genes are the NanoDrop Spectrophotometer (NanoDrop Technol- classified along three domains, namely molecular function ogies, Wilmington, DE, USA) and the Agilent 2100 (i.e. activity of gene products), cellular component (i.e. site Bioanalyzer (Agilent Technologies). All samples had an of active gene product) and biological process (i.e. path- RNA integrity number above 7.5. ways comprising the activities of multiple gene pro- ducts), and each domain is further sub-divided into a cDNA synthesis and quantitative real-time PCR hierarchical structure. In addition, each GO annotated cDNA synthesis and quantitative real-time-PCR (qPCR) gene has a defined relationship to other genes within 26,32 were performed as previously described . Relative gene the same domain, or to other domains, indicating that 36,37 expression levels were determined by the comparative C there is some overlap between the three domains . method (ΔΔC ), using Acidic ribosomal phosphoprotein In order to reduce false-positive—and negative—find- P0 (Arbp) and β-actin (Actb) as endogenous controls. The ings, we implemented highly stringent analysis settings: two-sided Student´s t-test was used to assess statistical 25–200 genes per gene set, 5000 permutations and significance between groups (p-value < 0.05). FDR ≤ 25. Microarray experiments Analysis of leading edge genes The microarray experiment was performed using the “Leading edge” (LE) genes, i.e. the genes driving the Agilent Technologies Rat Gene Expression 4x44K v3 enrichment score in the GSEA method, were exported Microarray Kit (Agilent Technologies). One hundred and and explored through Qiagen´s Ingenuity Pathway Ana- fifty nanograms total RNA from each FMCx sample was lysis (IPA) (Qiagen, Redwood City, CA, USA). reversely transcribed, amplified and Cy3-labelled using the Agilent Low Input Quick Amp Labelling Kit, one- Results colour v.6.6. 1.65 μg of Cy3-labelled cRNA was hybridised High plasma concentrations of olanzapine to Agilent Rat Gene Expression 4x44K v3 Microarrays, Five days after a single injection of long-acting olanza- which were subsequently washed according to the man- pine formulation (100 mg/kg), high and therapeutically ufacturer’s instructions. The Rat Gene Expression 4x44K relevant olanzapine plasma concentrations were deter- v3 Microarrays contain 45,018 probes representing 26,930 mined, ranging between 66.1 and 229.1 nM in the animals rat genes, sourced from RefSeq Build 36.2, Ensembl (n = 10) (mean ± S.E.M: 119.7 ± 15.8 nM). Release 55, Unigene Build 177 and GenBank (January 2009). Fluorescent signal detection was performed by the Olanzapine exposure leads to few changes at the single Agilent Technologies Scanner G2505B, and the resulting gene level in global gene expression in rat fronto-medial images were processed by Agilent Feature Extraction cortex Software version 10.7. SAM was used to identify differentially expressed single genes in the global gene expression data in FMCx from Microarray data analysis olanzapine-exposed animals (n = 10), vs. controls (n = Signal intensities were imported into J-Express 2012 10). At the global level, two genes were found to display (Molmine, Bergen, Norway) . To minimise the effect of significant downregulation (FDR = 0); the neuropeptide external technical variables (e.g. RNA extraction, labelling precursor encoding gene Vgf, and the neuropeptide- Translational Psychiatry Ersland et al. Translational Psychiatry (2017) 7:1262 Page 4 of 10 Vgf expression in rat fronto-medial cortex Cort expression in rat fronto-medial cortex Vehicle Olanzapine Vehicle Olanzapine Vgf Cort Vehicle Olanzapine Fig. 1 Downregulation of genes in rat fronto-medial cortex in response to olanzapine exposure. a SAM of global gene expression in FMCx from rats exposed to olanzapine (n = 10), compared to control animals (n = 10), revealed downregulated expression for two genes, Vgf and Cort. The box plots illustrates the relative expression levels (quantile normalised, log2-transformed singal intensities) of Vgf (left) and Cort (right), where the median of the distribution (thick black line), 75th percentile (upper edge of box), 25th percentile (lower edge of box), 95th percentile (upper edge of vertical line), 5th percentile (lower edge of vertical line) and the outlier points (above and below vertical lines) are indicated. b qPCR analysis revealed downregulated Vgf and Cort expression in response to olanzapine exposure (fold change: −1,43 and −1,47; p-values: 0.0013 and 0.004 (two-tailed Student's t-test), respectively). The y-axis indicates fold change relative to control animals. Genes are placed on the x-axis; samples from control animals (n = 10) (vehicle) in black, samples from olanzapine-exposed animals in grey (n = 10). Data are given as mean ± S.E.M. encoding gene Cort (fold change −1.25, and −1.48, We further examined whether the two neuropeptide- respectively) (Fig. 1a; for gene expression profiles across encoding genes displayed a similar downregulation in all samples, see Supplementary Figure 1). The differential three additional brain regions, namely hippocampus, gene expression was verified by qPCR (fold change: −1,43 striatum and hypothalamus. Using qPCR analysis, we and −1,47, p-value: 0.0013 and 0.004, for Vgf and Cort, found that Vgf, but not Cort, was downregulated in hip- respectively) (Fig. 1b). Of note, the third lowest FDR value pocampus in response to olanzapine exposure (fold in the SAM analysis was 70, far from the defined sig- change: −1,91, p-value: 0.017, data not shown). None of nificance threshold (Supplementary Table 1). the two genes were differentially expressed in striatum or hypothalamus. Translational Psychiatry QN log2 signal QN log2 signal Fold change relative to vehicle Ersland et al. Translational Psychiatry (2017) 7:1262 Page 5 of 10 Table 1 Gene set enrichment analysis of fronto-medial cortex in animals exposed to olanzapine, compared to control rats Gene set # p- FDR Leading edge genes Genes val Upregulated Ensheathment of neurons 59 0.00 13,1 n = 23, Cd9, Cldn11, Cntnap1, Gal3st1, Gjc3, Ilk, Jam3, Kel, Lpar1, Mal, Mtmr2, Nab1, Olig2, Pllp, Plp1, Pmp22, Pou3f1, Qki, Scd1, Serinc5, Tspan2, Ugt8, Xk Axon ensheathment 59 0.00 8,8 n = 23, Cd9, Cldn11, Cntnap1, Gal3st1, Gjc3, Ilk, Jam3, Kel, Lpar1, Mal, Mtmr2, Nab1, Olig2, Pllp, Plp1, Pmp22, Pou3f1, Qki, Scd1, Serinc5, Tspan2, Ugt8, Xk Myelination 57 0.00 19,1 n = 22, Cd9, Cldn11, Gal3st1, Gjc3, Ilk, Jam3, Kel, Lpar1, Mal, Mtmr2, Nab1, Olig2, Pllp, Plp1, Pmp22, Pou3f1, Qki, Scd1, Serinc5, Tspan2, Ugt8, Xk Myelin sheath 48 0.01 20,3 n = 16, Car2, Cnp, Gjc3, Hrh3, Itpr2, Itpr3, Jam3, Mag, Ncmap, Pllp, Plp1, Pmp22, Scrib, Serinc5, Sirt2, Tspan2 Oligodendrocyte 33 0.00 22,6 n = 8, Cd9, Fa2h, Gsn, Gstp1, Hdac10, Myrf, Nkx6-2, Plp1 development Downregulated Protein localisation in 26 0.00 17,3 n = 14, Ankrd13c, Bcap31, Kdelr1, Kdelr3, Macf1, Os9, Ryr2, Sec16b, Sec61a1, Srp19, Srp54a, Srp9, Srpr, endoplasmic reticulum Ubac2 Significance threshold was set to FDR < 25. Gene sets were derived from Gene Ontology (GO), organised with “ancestor” gene sets listed first, followed by “children” when present. p-val: nominal p-value, where a p-val = 0.00 indicates a p-value less than 1/5000 (one divided by the number of permutations performed). FDR, false discovery rate. Number of LE gene members in each gene set is listed in the Leading edge genes column. Genes highlighted in bold indicates the top five ranked genes from each gene set, in addition to the overlapping genes, analysed by qPCR. Myelination-related genes are upregulated at the gene-set ensheathment” and “Myelination” were found to be level in rat fronto-medial cortex in response to olanzapine identical, and subsequent analysis was performed using exposure the “Ensheathment of neurons” term (n = 23 genes) to GSEA was performed to identify potential sets of genes represent the three sets of genes. The LE genes from the with altered expression in response to olanzapine expo- two remaining gene sets, “Myelin sheath” (n = 16 genes) sure. This method is useful in detecting moderate and “Oligodendrocyte development” (n = 8 genes), did cumulative changes, in genes with an a priori defined not share any similarity, except for one gene, Plp1, which relationship (e.g. based on GO terms), which taken was common between all three gene sets (Fig. 2). The together can explain changes in expression that would not most overlap was found between the “Ensheathment of reach a conservative significance threshold in single gene neurons” and the “Myelin sheath” gene sets, sharing a total analysis. Under highly stringent settings (see Materials of six genes. and methods), a total of 1964 GO predefined gene sets Although none of the LE genes from the myelination- were tested, resulting in six gene sets identified as sig- related gene sets identified through the GSEA method nificantly changed in FMCx in response to olanzapine were found to be significantly different in the single gene- exposure (Table 1). Five of these gene sets were upregu- based SAM analysis (see above) many of them were lated in olanzapine-exposed animals, and strikingly, all of indeed upregulated upon independent re-examination. them were linked to oligodendrocytes and myelination We analysed a subset of these genes by qPCR, i.e. the top processes; “Ensheathment of neurons” (FDR = 13,1), five LE genes from each gene set, in addition to the LE “Axon ensheathment” (FDR = 8,8), “Myelination” (FDR = genes showing overlap between the three gene sets, 19,1), “Myelin sheath” (FDR = 20,3) and “Oligodendrocyte resulting in a total of 16 unique genes (highlighted in bold development” (FDR = 22,6) (Table 1). The genes with a in Table 1 and listed in Supplementary Table 3). Of these, positive contribution to the enrichment score (i.e. the we were able to show significant upregulation of 11 genes “leading edge” (LE) genes) for each gene set (Supple- (Fig. 3), namely Plp1, Serinc5, Tspan2, Fa2h, Mag, Mal, mentary Table 2) were further explored. The LE genes for Gjc3, Gstp1, Nkx6.2, Pllp and Cnp. Two other genes, Cd9 the gene sets “Ensheathment of neurons”, “Axon and Jam, displayed a trend towards upregulation (p- Translational Psychiatry Ersland et al. Translational Psychiatry (2017) 7:1262 Page 6 of 10 Cldn11, Cntnap1, Gjc3, Jam3, Ca2, Cnp, Hrh3, Gal3st1, Ilk, Kel, Pllp, Pmp22, Itpr2, Itpr3, Mag, Lpar1, Mal, Mtmr2, Serinc5, Tspan2 Ncmap, Scrib, Sirt2 Nab1, Olig2, Pou3f1, (n=6) (n=9) Ensheathment Myelin sheath Scd1, Qki, Ugt8, Xk of neurons (n=15) Plp1 Cd9 Fa2h, Gsn, Gstp1, Hdac10, Myrf, Nkx6-2 (n=6) Oligodendrocyte development Fig. 2 Overlap between myelination-related gene sets. Venn diagram illustrating the overlap between leading edge (LE) genes from the three gene sets “Ensheathment of neurons”, “Myelin sheath” and “Oligodendrocyte development”. The gene symbol and number of overlapping genes, from each gene set, are listed in the intersection between gene sets. 0 indicates no overlapping genes. The figure was generated by Ingenuity Pathway Analysis (IPA) tool In the GSEA, only one gene set was found to be ** ** downregulated in the FMCx, and this gene set was linked ** to translational processes (“Protein localisation in endo- plasmic reticulum”, Table 1). ** Effect of olanzapine exposure on myelination-related genes in hippocampus, striatum and hypothalamus Five of the LE genes ranked as most significant in the three genes sets, namely Serinc5, Tspan2, Cnp, Fa2h and Plp1, were further analysed by qPCR in samples from hippocampus, striatum and hypothalamus. Only one gene, Serinc5, was found to display a similar upregulation n.d. n.d. in the hippocampus as in FMCx (fold change: 1,23, p-val: 0,023). No differential gene expression was observed in the hippocampus for the four other genes in response to Vehicle Olanzapine olanzapine exposure. In striatum and hypothalamus, none of the selected LE genes displayed differential gene Fig. 3 qPCR analysis of “Leading Edge” genes. Sixteen LE genes, selected based on their rank in the gene set, and overlap between expression patterns in response to olanzapine exposure gene sets, were analysed by qPCR. Eleven out of the selected LE genes (data not shown). were significantly upregulated in response to olanzapine exposure in the FMCx. Gene symbols are listed at the x-axis, while the y-axis Discussion indicates fold change relative to control animals. Samples from control In this study we analysed alterations in global gene animals (n = 10) (vehicle) in black, samples from olanzapine-exposed animals (n = 10) in grey. Data are given as mean ± S.E.M. *: p-val < 0.05, expression in the rat fronto-medial cortex, induced by **: p-val < 0.01, n.d.: not detected, as determined using two-tailed olanzapine at stable and clinically relevant serum con- Student's t-test centrations. A marked upregulation of genes linked to oligodendrocytes and myelination processes was identi- fied, in addition to changes in two neuropeptide-encoding values: 0,057 and 0,093, respectively). The remaining three genes. genes were either not detected (i.e. Gal3st1 and Myrf), or Of the ~2000 GO predefined gene sets analysed by did not reach the significance threshold (i.e. Pmp22). GSEA, we observed an upregulation of five gene sets, all of Translational Psychiatry Cd9 Cnp Fah2 Gal3st1 Gjc3 Gstp1 Jam3 Mag Mal Myrf Nkx6_1 Pllp Plp1 Pmp22 Serinc5 Tspan2 Fold change relative to vehicle Ersland et al. Translational Psychiatry (2017) 7:1262 Page 7 of 10 which were linked to oligodendrocyte development and four transmembrane domains), i.e. Tspan2, Mal, Pllp, myelination. Three of the gene sets shared the same LE Cd9, Pmp22 and Plp1. Such tetraspans constitute a large genes, and were thus treated as similar, resulting in a total fraction of myelin proteins, and have a role in formation of three different myelination-related gene sets, namely of membrane junctions and regulation of growth and “Ensheathment of neurons”, “Myelin sheath” and “Oligo- migration of myelin-producing cells . Plp1, the only LE dendrocyte development”. Both “Ensheathment of neu- gene found in all three gene sets, has previously been rons” and “Oligodendrocyte development” belong to the shown to display almost exclusive expression in newly GO defined biological process domain, while “Myelin formed- and mature myelinating oligodendrocytes, con- sheath” is classified as belonging to the cellular compo- stituting the most abundant myelin tetraspan protein in 48–50 nent domain. Myelination has received increasing atten- the central nervous system . The encoded protein has tion in schizophrenia research over the last decades. Post- an important role in the biogenesis and structure of mortem-, but also imaging- and genetic studies, have myelin and maintenance of myelin sheaths, in addition to provided evidence for abnormalities in white matter and oligodendrocyte development and axonal survival . The 38–45 myelin . One of the most consistent findings from third gene set found to be upregulated in response to global gene expression studies in post-mortem brain olanzapine in the FMCx, “Oligodendrocyte development”, samples from patients suffering from schizophrenia has comprised mainly LE genes encoding intracellular pro- been downregulation of genes related to normal oligo- teins, such as the transcription factor Nkx6.2. This factor 12–16 13 dendrocyte function and myelination . Hakak et al. has previously been implicated in regulation of the mye- were the first to report reduced expression of lination process, being expressed in differentiating, but 50,52 myelination-related genes in the dorsolateral prefrontal also in newly formed and mature oligodendrocytes .It cortex from elderly schizophrenic patients. Interestingly, has multiple binding sites in the promoter regions of out of the six genes reported to be downregulated by Mbp and Plp1, further supporting that this factor Hakak et al., four were among the upregulated LE genes could be involved in regulating myelin-specific gene 52,53 identified in our analysis (i.e. Mag, Mal, Cnp and Gsn). In expression . a different study, three genes were found to be down- The transcriptional upregulation of genes linked to regulated in the temporal cortex of patients suffering from oligodendrocyte development and structural components schizophrenia, and two of these were among the LE genes of myelin in response to olanzapine exposure could sug- identified by us, namely Mal and Pllp . gest that one of olanzapine’s antipsychotic actions is to Interestingly, a recent study found that compensate for abnormal myelination, e.g. by activating oligodendrocyte-related gene sets (based on literature dormant oligodendrocyte progenitor cells into mature studies and GO annotations, i.e. “lipid metabolism”, but myelin-producing oligodendrocytes and promote myelin also “oxidation-reduction”, and “gene transcription”) were integrity . We did not observe such an upregulation significantly associated to the risk of developing schizo- outside cortical areas in our studies, suggesting that this phrenia . Most of the genes comprising the “Lipid effect may be restricted to the cortex. In fact, only one metabolism” gene set were shown to be involved in gene, Serinc5, showed increased expression elsewhere, metabolism of structural membrane lipids of the myelin namely within the hippocampus. sheath, with additional roles in sorting, trafficking and Indeed, imaging studies have demonstrated increased anchoring of myelin proteins in the myelin membrane . white matter volume in the cerebral cortex in response to In our analysis, two of the upregulated gene sets, antipsychotic medication, primarily due to increased 55,56 “Ensheathment of neurons” and “Myelin sheath”, com- myelination at deeper cortical levels . Studies in ani- prised LE genes primarily linked to structural components mals receiving antipsychotic medications have also of the myelin sheath. Serinc5 was ranked as the most demonstrated morphological changes in the brain, enriched gene in both of these two gene sets. The encoded including increased glial density in deeper cortical lay- Serinc5 protein is an important factor in facilitating and ers . Furthermore, gene expression studies in mice have regulating biosynthesis of myelin glycolipids, which is a reported on alterations in lipid-related genes in response major component of myelin (27% of the total lipid con- to the antipsychotics clozapine and haloperidol in 47 58 tent) . The encoded Serinc5 protein localises to endo- cortex, but also to a certain extent in striatum . Finally, plasmatic reticulum membranes, where it binds directly to cuprizone-induced demyelination in mice can be lipid biosynthetic enzymes also embedded there, incor- prevented by exposure to the second-generation anti- porating the nonessential amino acid serine into glycoli- psychotic quetiapine, which also promoted oligoden- 47 59 pids synthesised in oligodendrocytes . In addition, the drocyte development . Although most work in the field “Ensheathment of neurons” gene set, and to a lesser extent has been focusing on quetiapine, one study also examined the “Myelin sheath” gene set, comprised several genes the effect of olanzapine in cuprizone-induced demyeli- encoding tetraspans (membrane-embedded proteins with nation in mice. Here, olanzapine was found to effectively Translational Psychiatry Ersland et al. Translational Psychiatry (2017) 7:1262 Page 8 of 10 decrease myelin breakdown and loss of oligodendrocytes, samples from patients diagnosed with schizophrenia. in response to cuprizone exposure in mice . Further Antipsychotic-induced induction of myelination-related studies should aim at resolving whether the olanzapine gene expression may be relevant both to the clinical induced upregulated expression of genes linked to oligo- effects of antipsychotic agents and to the pathophysiology dendrocytes and myelination processes is a general effect, of schizophrenia. by comparing different antipsychotic drugs head to head. Acknowledgements In addition, such studies should also determine whether The authors would like to thank Marianne Nævdal and Kjell Ove Fossan for the effect could be observed at several different time- excellent technical assistance. We also acknowledge the research infrastructure provided by the Genomics Core Facility (GCF) and the Laboratory animal points. Our study did not consider the potential con- facility (Vivarium), at the University of Bergen, Norway. The work was funded by founding effect of the oestrous cycle stage, which has a grant from the national health authorities (Helse Vest RHF, project-ID: previously been demonstrated to influence gene expres- 509035), the Research Council of Norway (NORMENT CoE; project no. 223,273) and Stiftelsen Kristian Gerhard Jebsen. sion in rat medial prefrontal cortex . However, none of the individual genes found to be differentially regulated in Author details our study seemed to be affected by oestrous stage . 1 Dr. Einar Martens Research Group for Biological Psychiatry, Center for Medical At the single gene level, we found that the two Genetics and Molecular Medicine, Haukeland University Hospital, Bergen 5021, Norway. The Norwegian Centre for Mental Disorders Research (NORMENT) neuropeptide-encoding genes Vgf and Cort were sig- and the K.G. Jebsen Centre for Psychosis Research, Department of Clinical nificantly downregulated in the rat FMCx in response to Science, University of Bergen, Bergen, Norway olanzapine exposure. In the brain, Vgf is widely dis- tributed, but shows particular abundance in distinct areas, Competing interests The authors declare that they have no competing financial interests. such as the cerebral cortex, hippocampus, olfactory sys- 62,63 tem and hypothalamus . The encoded protein gives Publisher's note: Springer Nature remains neutral with regard to jurisdictional rise to several low molecular weight neuropeptides, and claims in published maps and institutional affiliations. 64,65 has been implicated in regulation of energy balance . Electronic supplementary material However, the exact function of the majority of Vgf neu- The online version of this article (https://doi.org/10.1038/s41398-017-0008-3) ropeptides remains elusive. In our study, expression of contains supplementary material, which is available to authorized users. Vgf was also downregulated in hippocampus, but no alterations were observed in the striatum or hypothala- Received: 3 March 2017 Accepted: 14 July 2017 mus. The second identified gene, Cort, is primarily expressed by GABAergic interneurons, and has a scat- tered expression pattern across the cerebral cortex .In References our study, this gene was downregulated in FMCx after 1. McGrath, J.,Saha, S.,Chant,D. & Welham, J. Schizophrenia: a concise overview olanzapine exposure. Stable expression levels within hip- of incidence, prevalence, and mortality. Epidemiol. Rev. 30,67–76 (2008). pocampus, striatum and hypothalamus could indicate a 2. Lichtenstein, P. et al. Common genetic determinants of schizophrenia and bipolar disorder in Swedish families: a population-based study. 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Subchronic olanzapine exposure leads to increased expression of myelination-related genes in rat fronto-medial cortex

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Medicine & Public Health; Medicine/Public Health, general; Psychiatry; Neurosciences; Behavioral Sciences; Pharmacotherapy; Biological Psychology
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Abstract

Schizophrenia is a psychotic disorder with severe and disabling symptoms, such as hallucinations, delusions, blunted affect and social withdrawal. The neuropathology remains elusive, but disturbances in immunity-related processes, neuronal connectivity and myelination have consistently been linked to schizophrenia. Antipsychotic drugs can be efficient in reducing symptoms, acting primarily on the dopamine system, but additional biological targets are likely to exist. Here we have screened for novel mechanisms of action in an animal model, using adult rats exposed to long- acting olanzapine, achieving stable and clinically relevant antipsychotic drug concentrations. By microarray-based examination of global gene expression in the fronto-medial cortex, at the single gene- and gene-set level, we observed downregulation of two neuropeptide-encoding genes, Vgf and Cort (fold change −1,25 and −1,48, respectively) in response to olanzapine exposure. Furthermore, we demonstrated significant upregulation of five out of ~2000 GO predefined gene sets after olanzapine exposure. Strikingly, all were linked to myelination and oligodendrocyte development; “Ensheathment of neurons”, “Axon ensheathment”, “Myelination”, “Myelin sheath” and “Oligodendrocyte development” (FDR-values < 25). Sixteen of the leading edge genes in these gene sets were analysed independently by qPCR, of which 11 genes displayed significant upregulation, including Plp1, Mal, Mag and Cnp (fold change: 1,30, 1,50, 1,30 and 1,15, respectively). Several of the upregulated genes (e.g. MAG, MAL and CNP) have previously been reported as downregulated in post-mortem brain samples from schizophrenia patients. Although caution needs to be taken when extrapolating results from animal studies to humans, the data suggest a role for olanzapine in alleviating myelination-related dysfunction in schizophrenia. Introduction maturation, including myelination. Although the clinical Schizophrenia is a disabling psychiatric disorder with a manifestations have been known for centuries, the lifetime prevalence of about 0.7% . The illness is char- underlying pathophysiology and aetiology of the disorder acterised by complex clinical symptoms, including hallu- remain largely unknown. While the estimated heritability 2,3 cinations, delusions, social withdrawal and blunted affect, ranges from 60 to 80% , and recent studies have started 4–8 in addition to cognitive impairment. The onset of schi- to disclose genetic risk factors of schizophrenia , much zophrenia is normally during late adolescence or early still remains to be elucidated. adulthood, a time period coinciding with brain As an alternative approach to study the pathological disease mechanisms, large-scale gene expression studies have been performed on post-mortem brain samples from Correspondence: Kari M Ersland (kari.ersland@uib.no) patients suffering from schizophrenia. The most con- Dr. Einar Martens Research Group for Biological Psychiatry, Center for Medical sistent observations from these studies include alterations Genetics and Molecular Medicine, Haukeland University Hospital, Bergen 5021, in expression levels for genes related to brain white matter Norway The Norwegian Centre for Mental Disorders Research (NORMENT) and the K.G. and oligodendrocytes, signalling and synapses, GABA and Jebsen Centre for Psychosis Research, Department of Clinical Science, glutamate neurotransmission, mitochondrial function, in University of Bergen, Bergen, Norway © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Translational Psychiatry 1234567890 1234567890 Ersland et al. Translational Psychiatry (2017) 7:1262 Page 2 of 10 9–11 29,30 addition to immune and stress-response (reviewed in ). stimulating effects . We demonstrate a significant In particular, several studies have reported a down- downregulation of two neuropeptide-encoding genes, and regulation of genes linked to myelination and myelin- interestingly, a striking upregulation of gene sets linked to 12–16 producing oligodendrocytes in schizophrenia . Oligo- myelination-related functional pathways. To our knowl- dendrocyte membranes wrap around axons forming a edge, this is the first study to report on global gene multi-lamellar myelin structure, which is essential for expression alterations in rat brain in response to stable rapid propagation of action potentials and the long-term and clinically relevant olanzapine levels. integrity of neuronal axons. Since myelin is a lipid-rich membrane structure, it is interesting that several anti- Materials and methods psychotic drugs, especially clozapine and olanzapine, have Animals and drug exposure scheme been shown to have lipid-stimulating effects. The experiment was approved by, and carried out in The results from post-mortem brain studies have, accordance with the guidelines of the Norwegian Com- however, been challenging to replicate, probably attrib- mittee for Experiments on Animals (Forsøksdyrutvalget, uted to cofounding factors, e.g. age, severity of disease, FDU), following standardised application through the treatment with antipsychotic drugs, co-morbidity, animal facility at Haukeland University Hospital (ID post-mortem sample handling and type of microarray 2014–6735). Ten to 12 weeks old female outbred Sprague- platform used . Obstacles such as these are less promi- Dawley rats (weighing ~240 g) (Mollegaard, Denmark) nent when using animals to explore drug-induced were housed under standard conditions with an artificial changes in gene expression in the brain, although such 12:12 h light/dark cycle under constant 48% humidity. models have obvious limitations in the study of psychia- Female rats were selected as the administration of long- tric disorders . Rodents, primarily rats, have been acting olanzapine has resulted in clinically relevant extensively used to analyse the effect of various anti- metabolic phenotypes, while exposure of male rats have 26–28,31 psychotic drugs on gene expression in different areas of yielded less clear-cut results . Five animals were the brain. Overall, the alterations identified seem to housed in each cage, with free access to standard converge on some of the same pathways as observed in laboratory chow (Special Diets Services, Witham, UK) and patient post-mortem studies, including neurotransmis- tap water. Animals were randomly divided into two sion, neural plasticity, cell survival, ionic homoeostasis groups (n = 10) (the investigators were blinded to the 11,18–21 and synaptic functions . group allocations); the first group received a single Still, direct comparison between animal studies have intramuscular injection of commercially available olan- been problematic due to differences in methodology, zapine pamoate depot formulation (100 mg/kg BW brain areas examined, choice of antipsychotic drugs and ZypAdhera ) (Eli Lilly, Indianapolis, IN, USA), while the treatment schemes. The rapid metabolism of anti- second group received commercially available vehicle psychotic agents in rodents represents an additional solution (injection volume 160 μl/250 g BW) on the first challenge, impending clinically relevant serum con- day of the experimental period, as previously described . centrations of antipsychotic drugs, especially over time. Sample size and the injected dose were determined based 26,28 As an example, the half-life (t ) of the second-generation on previous experiments . Food was removed 10 h 1/2 antipsychotic olanzapine in rat serum is 3 h, while the before sacrifice, in order to reduce potential variability in 22–24 average t is 30 h in humans . Most global gene gene expression caused by differences in metabolic para- 1/2 expression studies to date have used daily oral adminis- meters. Based on previous rat experiments of gene tration of antipsychotic agents (mainly through drinking expression in peripheral tissues, a time period of 5 days 26,28 water, but also by gavage) or daily injections. Recently, our was selected, representing a sub-acute exposure time . group demonstrated that the use of depot injections of Care was taken to ensure minimal suffering of the animals atypical antipsychotics in rat leads to clinically relevant at all stages of the experiments. and stable serum concentrations over time, as compared to the fluctuating and often low serum drug concentra- Tissue dissection tions observed in rats exposed to twice-daily oral drug Rats were anesthetised by isoflurane gas (Isoba vet; 25–28 administration during the same time period . Schering-Plough, Denmark), and subsequently sacrificed In this work, we exposed rats to a 5-day treatment with by decapitation. Brains were removed from the skull, long-acting olanzapine to examine alterations in global washed in cold phosphate-buffered saline and placed on gene expression in the fronto-medial parts of the cerebral ice. Tissue samples from the fronto-medial part of the cortex, identifying drug-induced molecular profiles. We cortex (FMCx), striatum and hippocampus were dissected chose to study olanzapine, since this antipsychotic drug from the right hemisphere. In addition, the whole hypo- has been shown to be one of the most efficacious anti- thalamus was dissected. Tissue samples were immediately psychotic agents, in addition to displaying strong lipid- frozen on dry ice, and stored at −80 °C. Translational Psychiatry Ersland et al. Translational Psychiatry (2017) 7:1262 Page 3 of 10 Measurement of plasma olanzapine levels and hybridisation) and to obtain a normal distribution, Truncal blood was collected as previously described . inter-array quantile normalisation and log2 transforma- Plasma levels of olanzapine were determined by means of tion was performed, respectively. Identification of single high-performance liquid chromatography using Agilent microarray probes that differed significantly in expression 1290 Infinity Binary LC, coupled to an Agilent 6490A level (i.e. hybridisation signal intensity) between sample triple quadrupole mass spectrometer using positive elec- groups was carried out by significance analysis of micro- trospray ionisation (Agilent Technologies, Santa Clara, arrays (SAM) , comparing signal intensities of all probes CA, USA). across all samples. In order to reduce the number of false- positive findings, the significance threshold was set to a RNA extraction highly conservative false discovery rate (FDR) of 0 and Tissue samples from FMCx, hypothalamus, hippo- 1000 permutations were performed. Statistically sig- campus and striatum were homogenised using a Tissue- nificant gene expression profiles were further explored Lyser (Qiagen, Hilden, Germany). RNA extraction was and curated by manual inspection. Gene set enrichment performed on an ABI Prism™ 6100 Nucleic Acid Pre- analysis (GSEA) was used to analyse the global gene pStation (Applied Biosystems, Foster City, CA, USA), with expression data, at the gene-set level. A priori defined DNase treatment according to the manufacturer’s proto- gene sets were based on Gene Ontology (GO) annota- col. Amount and quality of total RNA was measured using tions, implemented in J-Express . In GO, genes are the NanoDrop Spectrophotometer (NanoDrop Technol- classified along three domains, namely molecular function ogies, Wilmington, DE, USA) and the Agilent 2100 (i.e. activity of gene products), cellular component (i.e. site Bioanalyzer (Agilent Technologies). All samples had an of active gene product) and biological process (i.e. path- RNA integrity number above 7.5. ways comprising the activities of multiple gene pro- ducts), and each domain is further sub-divided into a cDNA synthesis and quantitative real-time PCR hierarchical structure. In addition, each GO annotated cDNA synthesis and quantitative real-time-PCR (qPCR) gene has a defined relationship to other genes within 26,32 were performed as previously described . Relative gene the same domain, or to other domains, indicating that 36,37 expression levels were determined by the comparative C there is some overlap between the three domains . method (ΔΔC ), using Acidic ribosomal phosphoprotein In order to reduce false-positive—and negative—find- P0 (Arbp) and β-actin (Actb) as endogenous controls. The ings, we implemented highly stringent analysis settings: two-sided Student´s t-test was used to assess statistical 25–200 genes per gene set, 5000 permutations and significance between groups (p-value < 0.05). FDR ≤ 25. Microarray experiments Analysis of leading edge genes The microarray experiment was performed using the “Leading edge” (LE) genes, i.e. the genes driving the Agilent Technologies Rat Gene Expression 4x44K v3 enrichment score in the GSEA method, were exported Microarray Kit (Agilent Technologies). One hundred and and explored through Qiagen´s Ingenuity Pathway Ana- fifty nanograms total RNA from each FMCx sample was lysis (IPA) (Qiagen, Redwood City, CA, USA). reversely transcribed, amplified and Cy3-labelled using the Agilent Low Input Quick Amp Labelling Kit, one- Results colour v.6.6. 1.65 μg of Cy3-labelled cRNA was hybridised High plasma concentrations of olanzapine to Agilent Rat Gene Expression 4x44K v3 Microarrays, Five days after a single injection of long-acting olanza- which were subsequently washed according to the man- pine formulation (100 mg/kg), high and therapeutically ufacturer’s instructions. The Rat Gene Expression 4x44K relevant olanzapine plasma concentrations were deter- v3 Microarrays contain 45,018 probes representing 26,930 mined, ranging between 66.1 and 229.1 nM in the animals rat genes, sourced from RefSeq Build 36.2, Ensembl (n = 10) (mean ± S.E.M: 119.7 ± 15.8 nM). Release 55, Unigene Build 177 and GenBank (January 2009). Fluorescent signal detection was performed by the Olanzapine exposure leads to few changes at the single Agilent Technologies Scanner G2505B, and the resulting gene level in global gene expression in rat fronto-medial images were processed by Agilent Feature Extraction cortex Software version 10.7. SAM was used to identify differentially expressed single genes in the global gene expression data in FMCx from Microarray data analysis olanzapine-exposed animals (n = 10), vs. controls (n = Signal intensities were imported into J-Express 2012 10). At the global level, two genes were found to display (Molmine, Bergen, Norway) . To minimise the effect of significant downregulation (FDR = 0); the neuropeptide external technical variables (e.g. RNA extraction, labelling precursor encoding gene Vgf, and the neuropeptide- Translational Psychiatry Ersland et al. Translational Psychiatry (2017) 7:1262 Page 4 of 10 Vgf expression in rat fronto-medial cortex Cort expression in rat fronto-medial cortex Vehicle Olanzapine Vehicle Olanzapine Vgf Cort Vehicle Olanzapine Fig. 1 Downregulation of genes in rat fronto-medial cortex in response to olanzapine exposure. a SAM of global gene expression in FMCx from rats exposed to olanzapine (n = 10), compared to control animals (n = 10), revealed downregulated expression for two genes, Vgf and Cort. The box plots illustrates the relative expression levels (quantile normalised, log2-transformed singal intensities) of Vgf (left) and Cort (right), where the median of the distribution (thick black line), 75th percentile (upper edge of box), 25th percentile (lower edge of box), 95th percentile (upper edge of vertical line), 5th percentile (lower edge of vertical line) and the outlier points (above and below vertical lines) are indicated. b qPCR analysis revealed downregulated Vgf and Cort expression in response to olanzapine exposure (fold change: −1,43 and −1,47; p-values: 0.0013 and 0.004 (two-tailed Student's t-test), respectively). The y-axis indicates fold change relative to control animals. Genes are placed on the x-axis; samples from control animals (n = 10) (vehicle) in black, samples from olanzapine-exposed animals in grey (n = 10). Data are given as mean ± S.E.M. encoding gene Cort (fold change −1.25, and −1.48, We further examined whether the two neuropeptide- respectively) (Fig. 1a; for gene expression profiles across encoding genes displayed a similar downregulation in all samples, see Supplementary Figure 1). The differential three additional brain regions, namely hippocampus, gene expression was verified by qPCR (fold change: −1,43 striatum and hypothalamus. Using qPCR analysis, we and −1,47, p-value: 0.0013 and 0.004, for Vgf and Cort, found that Vgf, but not Cort, was downregulated in hip- respectively) (Fig. 1b). Of note, the third lowest FDR value pocampus in response to olanzapine exposure (fold in the SAM analysis was 70, far from the defined sig- change: −1,91, p-value: 0.017, data not shown). None of nificance threshold (Supplementary Table 1). the two genes were differentially expressed in striatum or hypothalamus. Translational Psychiatry QN log2 signal QN log2 signal Fold change relative to vehicle Ersland et al. Translational Psychiatry (2017) 7:1262 Page 5 of 10 Table 1 Gene set enrichment analysis of fronto-medial cortex in animals exposed to olanzapine, compared to control rats Gene set # p- FDR Leading edge genes Genes val Upregulated Ensheathment of neurons 59 0.00 13,1 n = 23, Cd9, Cldn11, Cntnap1, Gal3st1, Gjc3, Ilk, Jam3, Kel, Lpar1, Mal, Mtmr2, Nab1, Olig2, Pllp, Plp1, Pmp22, Pou3f1, Qki, Scd1, Serinc5, Tspan2, Ugt8, Xk Axon ensheathment 59 0.00 8,8 n = 23, Cd9, Cldn11, Cntnap1, Gal3st1, Gjc3, Ilk, Jam3, Kel, Lpar1, Mal, Mtmr2, Nab1, Olig2, Pllp, Plp1, Pmp22, Pou3f1, Qki, Scd1, Serinc5, Tspan2, Ugt8, Xk Myelination 57 0.00 19,1 n = 22, Cd9, Cldn11, Gal3st1, Gjc3, Ilk, Jam3, Kel, Lpar1, Mal, Mtmr2, Nab1, Olig2, Pllp, Plp1, Pmp22, Pou3f1, Qki, Scd1, Serinc5, Tspan2, Ugt8, Xk Myelin sheath 48 0.01 20,3 n = 16, Car2, Cnp, Gjc3, Hrh3, Itpr2, Itpr3, Jam3, Mag, Ncmap, Pllp, Plp1, Pmp22, Scrib, Serinc5, Sirt2, Tspan2 Oligodendrocyte 33 0.00 22,6 n = 8, Cd9, Fa2h, Gsn, Gstp1, Hdac10, Myrf, Nkx6-2, Plp1 development Downregulated Protein localisation in 26 0.00 17,3 n = 14, Ankrd13c, Bcap31, Kdelr1, Kdelr3, Macf1, Os9, Ryr2, Sec16b, Sec61a1, Srp19, Srp54a, Srp9, Srpr, endoplasmic reticulum Ubac2 Significance threshold was set to FDR < 25. Gene sets were derived from Gene Ontology (GO), organised with “ancestor” gene sets listed first, followed by “children” when present. p-val: nominal p-value, where a p-val = 0.00 indicates a p-value less than 1/5000 (one divided by the number of permutations performed). FDR, false discovery rate. Number of LE gene members in each gene set is listed in the Leading edge genes column. Genes highlighted in bold indicates the top five ranked genes from each gene set, in addition to the overlapping genes, analysed by qPCR. Myelination-related genes are upregulated at the gene-set ensheathment” and “Myelination” were found to be level in rat fronto-medial cortex in response to olanzapine identical, and subsequent analysis was performed using exposure the “Ensheathment of neurons” term (n = 23 genes) to GSEA was performed to identify potential sets of genes represent the three sets of genes. The LE genes from the with altered expression in response to olanzapine expo- two remaining gene sets, “Myelin sheath” (n = 16 genes) sure. This method is useful in detecting moderate and “Oligodendrocyte development” (n = 8 genes), did cumulative changes, in genes with an a priori defined not share any similarity, except for one gene, Plp1, which relationship (e.g. based on GO terms), which taken was common between all three gene sets (Fig. 2). The together can explain changes in expression that would not most overlap was found between the “Ensheathment of reach a conservative significance threshold in single gene neurons” and the “Myelin sheath” gene sets, sharing a total analysis. Under highly stringent settings (see Materials of six genes. and methods), a total of 1964 GO predefined gene sets Although none of the LE genes from the myelination- were tested, resulting in six gene sets identified as sig- related gene sets identified through the GSEA method nificantly changed in FMCx in response to olanzapine were found to be significantly different in the single gene- exposure (Table 1). Five of these gene sets were upregu- based SAM analysis (see above) many of them were lated in olanzapine-exposed animals, and strikingly, all of indeed upregulated upon independent re-examination. them were linked to oligodendrocytes and myelination We analysed a subset of these genes by qPCR, i.e. the top processes; “Ensheathment of neurons” (FDR = 13,1), five LE genes from each gene set, in addition to the LE “Axon ensheathment” (FDR = 8,8), “Myelination” (FDR = genes showing overlap between the three gene sets, 19,1), “Myelin sheath” (FDR = 20,3) and “Oligodendrocyte resulting in a total of 16 unique genes (highlighted in bold development” (FDR = 22,6) (Table 1). The genes with a in Table 1 and listed in Supplementary Table 3). Of these, positive contribution to the enrichment score (i.e. the we were able to show significant upregulation of 11 genes “leading edge” (LE) genes) for each gene set (Supple- (Fig. 3), namely Plp1, Serinc5, Tspan2, Fa2h, Mag, Mal, mentary Table 2) were further explored. The LE genes for Gjc3, Gstp1, Nkx6.2, Pllp and Cnp. Two other genes, Cd9 the gene sets “Ensheathment of neurons”, “Axon and Jam, displayed a trend towards upregulation (p- Translational Psychiatry Ersland et al. Translational Psychiatry (2017) 7:1262 Page 6 of 10 Cldn11, Cntnap1, Gjc3, Jam3, Ca2, Cnp, Hrh3, Gal3st1, Ilk, Kel, Pllp, Pmp22, Itpr2, Itpr3, Mag, Lpar1, Mal, Mtmr2, Serinc5, Tspan2 Ncmap, Scrib, Sirt2 Nab1, Olig2, Pou3f1, (n=6) (n=9) Ensheathment Myelin sheath Scd1, Qki, Ugt8, Xk of neurons (n=15) Plp1 Cd9 Fa2h, Gsn, Gstp1, Hdac10, Myrf, Nkx6-2 (n=6) Oligodendrocyte development Fig. 2 Overlap between myelination-related gene sets. Venn diagram illustrating the overlap between leading edge (LE) genes from the three gene sets “Ensheathment of neurons”, “Myelin sheath” and “Oligodendrocyte development”. The gene symbol and number of overlapping genes, from each gene set, are listed in the intersection between gene sets. 0 indicates no overlapping genes. The figure was generated by Ingenuity Pathway Analysis (IPA) tool In the GSEA, only one gene set was found to be ** ** downregulated in the FMCx, and this gene set was linked ** to translational processes (“Protein localisation in endo- plasmic reticulum”, Table 1). ** Effect of olanzapine exposure on myelination-related genes in hippocampus, striatum and hypothalamus Five of the LE genes ranked as most significant in the three genes sets, namely Serinc5, Tspan2, Cnp, Fa2h and Plp1, were further analysed by qPCR in samples from hippocampus, striatum and hypothalamus. Only one gene, Serinc5, was found to display a similar upregulation n.d. n.d. in the hippocampus as in FMCx (fold change: 1,23, p-val: 0,023). No differential gene expression was observed in the hippocampus for the four other genes in response to Vehicle Olanzapine olanzapine exposure. In striatum and hypothalamus, none of the selected LE genes displayed differential gene Fig. 3 qPCR analysis of “Leading Edge” genes. Sixteen LE genes, selected based on their rank in the gene set, and overlap between expression patterns in response to olanzapine exposure gene sets, were analysed by qPCR. Eleven out of the selected LE genes (data not shown). were significantly upregulated in response to olanzapine exposure in the FMCx. Gene symbols are listed at the x-axis, while the y-axis Discussion indicates fold change relative to control animals. Samples from control In this study we analysed alterations in global gene animals (n = 10) (vehicle) in black, samples from olanzapine-exposed animals (n = 10) in grey. Data are given as mean ± S.E.M. *: p-val < 0.05, expression in the rat fronto-medial cortex, induced by **: p-val < 0.01, n.d.: not detected, as determined using two-tailed olanzapine at stable and clinically relevant serum con- Student's t-test centrations. A marked upregulation of genes linked to oligodendrocytes and myelination processes was identi- fied, in addition to changes in two neuropeptide-encoding values: 0,057 and 0,093, respectively). The remaining three genes. genes were either not detected (i.e. Gal3st1 and Myrf), or Of the ~2000 GO predefined gene sets analysed by did not reach the significance threshold (i.e. Pmp22). GSEA, we observed an upregulation of five gene sets, all of Translational Psychiatry Cd9 Cnp Fah2 Gal3st1 Gjc3 Gstp1 Jam3 Mag Mal Myrf Nkx6_1 Pllp Plp1 Pmp22 Serinc5 Tspan2 Fold change relative to vehicle Ersland et al. Translational Psychiatry (2017) 7:1262 Page 7 of 10 which were linked to oligodendrocyte development and four transmembrane domains), i.e. Tspan2, Mal, Pllp, myelination. Three of the gene sets shared the same LE Cd9, Pmp22 and Plp1. Such tetraspans constitute a large genes, and were thus treated as similar, resulting in a total fraction of myelin proteins, and have a role in formation of three different myelination-related gene sets, namely of membrane junctions and regulation of growth and “Ensheathment of neurons”, “Myelin sheath” and “Oligo- migration of myelin-producing cells . Plp1, the only LE dendrocyte development”. Both “Ensheathment of neu- gene found in all three gene sets, has previously been rons” and “Oligodendrocyte development” belong to the shown to display almost exclusive expression in newly GO defined biological process domain, while “Myelin formed- and mature myelinating oligodendrocytes, con- sheath” is classified as belonging to the cellular compo- stituting the most abundant myelin tetraspan protein in 48–50 nent domain. Myelination has received increasing atten- the central nervous system . The encoded protein has tion in schizophrenia research over the last decades. Post- an important role in the biogenesis and structure of mortem-, but also imaging- and genetic studies, have myelin and maintenance of myelin sheaths, in addition to provided evidence for abnormalities in white matter and oligodendrocyte development and axonal survival . The 38–45 myelin . One of the most consistent findings from third gene set found to be upregulated in response to global gene expression studies in post-mortem brain olanzapine in the FMCx, “Oligodendrocyte development”, samples from patients suffering from schizophrenia has comprised mainly LE genes encoding intracellular pro- been downregulation of genes related to normal oligo- teins, such as the transcription factor Nkx6.2. This factor 12–16 13 dendrocyte function and myelination . Hakak et al. has previously been implicated in regulation of the mye- were the first to report reduced expression of lination process, being expressed in differentiating, but 50,52 myelination-related genes in the dorsolateral prefrontal also in newly formed and mature oligodendrocytes .It cortex from elderly schizophrenic patients. Interestingly, has multiple binding sites in the promoter regions of out of the six genes reported to be downregulated by Mbp and Plp1, further supporting that this factor Hakak et al., four were among the upregulated LE genes could be involved in regulating myelin-specific gene 52,53 identified in our analysis (i.e. Mag, Mal, Cnp and Gsn). In expression . a different study, three genes were found to be down- The transcriptional upregulation of genes linked to regulated in the temporal cortex of patients suffering from oligodendrocyte development and structural components schizophrenia, and two of these were among the LE genes of myelin in response to olanzapine exposure could sug- identified by us, namely Mal and Pllp . gest that one of olanzapine’s antipsychotic actions is to Interestingly, a recent study found that compensate for abnormal myelination, e.g. by activating oligodendrocyte-related gene sets (based on literature dormant oligodendrocyte progenitor cells into mature studies and GO annotations, i.e. “lipid metabolism”, but myelin-producing oligodendrocytes and promote myelin also “oxidation-reduction”, and “gene transcription”) were integrity . We did not observe such an upregulation significantly associated to the risk of developing schizo- outside cortical areas in our studies, suggesting that this phrenia . Most of the genes comprising the “Lipid effect may be restricted to the cortex. In fact, only one metabolism” gene set were shown to be involved in gene, Serinc5, showed increased expression elsewhere, metabolism of structural membrane lipids of the myelin namely within the hippocampus. sheath, with additional roles in sorting, trafficking and Indeed, imaging studies have demonstrated increased anchoring of myelin proteins in the myelin membrane . white matter volume in the cerebral cortex in response to In our analysis, two of the upregulated gene sets, antipsychotic medication, primarily due to increased 55,56 “Ensheathment of neurons” and “Myelin sheath”, com- myelination at deeper cortical levels . Studies in ani- prised LE genes primarily linked to structural components mals receiving antipsychotic medications have also of the myelin sheath. Serinc5 was ranked as the most demonstrated morphological changes in the brain, enriched gene in both of these two gene sets. The encoded including increased glial density in deeper cortical lay- Serinc5 protein is an important factor in facilitating and ers . Furthermore, gene expression studies in mice have regulating biosynthesis of myelin glycolipids, which is a reported on alterations in lipid-related genes in response major component of myelin (27% of the total lipid con- to the antipsychotics clozapine and haloperidol in 47 58 tent) . The encoded Serinc5 protein localises to endo- cortex, but also to a certain extent in striatum . Finally, plasmatic reticulum membranes, where it binds directly to cuprizone-induced demyelination in mice can be lipid biosynthetic enzymes also embedded there, incor- prevented by exposure to the second-generation anti- porating the nonessential amino acid serine into glycoli- psychotic quetiapine, which also promoted oligoden- 47 59 pids synthesised in oligodendrocytes . In addition, the drocyte development . Although most work in the field “Ensheathment of neurons” gene set, and to a lesser extent has been focusing on quetiapine, one study also examined the “Myelin sheath” gene set, comprised several genes the effect of olanzapine in cuprizone-induced demyeli- encoding tetraspans (membrane-embedded proteins with nation in mice. Here, olanzapine was found to effectively Translational Psychiatry Ersland et al. Translational Psychiatry (2017) 7:1262 Page 8 of 10 decrease myelin breakdown and loss of oligodendrocytes, samples from patients diagnosed with schizophrenia. in response to cuprizone exposure in mice . Further Antipsychotic-induced induction of myelination-related studies should aim at resolving whether the olanzapine gene expression may be relevant both to the clinical induced upregulated expression of genes linked to oligo- effects of antipsychotic agents and to the pathophysiology dendrocytes and myelination processes is a general effect, of schizophrenia. by comparing different antipsychotic drugs head to head. Acknowledgements In addition, such studies should also determine whether The authors would like to thank Marianne Nævdal and Kjell Ove Fossan for the effect could be observed at several different time- excellent technical assistance. We also acknowledge the research infrastructure provided by the Genomics Core Facility (GCF) and the Laboratory animal points. Our study did not consider the potential con- facility (Vivarium), at the University of Bergen, Norway. The work was funded by founding effect of the oestrous cycle stage, which has a grant from the national health authorities (Helse Vest RHF, project-ID: previously been demonstrated to influence gene expres- 509035), the Research Council of Norway (NORMENT CoE; project no. 223,273) and Stiftelsen Kristian Gerhard Jebsen. sion in rat medial prefrontal cortex . However, none of the individual genes found to be differentially regulated in Author details our study seemed to be affected by oestrous stage . 1 Dr. Einar Martens Research Group for Biological Psychiatry, Center for Medical At the single gene level, we found that the two Genetics and Molecular Medicine, Haukeland University Hospital, Bergen 5021, Norway. The Norwegian Centre for Mental Disorders Research (NORMENT) neuropeptide-encoding genes Vgf and Cort were sig- and the K.G. Jebsen Centre for Psychosis Research, Department of Clinical nificantly downregulated in the rat FMCx in response to Science, University of Bergen, Bergen, Norway olanzapine exposure. In the brain, Vgf is widely dis- tributed, but shows particular abundance in distinct areas, Competing interests The authors declare that they have no competing financial interests. such as the cerebral cortex, hippocampus, olfactory sys- 62,63 tem and hypothalamus . The encoded protein gives Publisher's note: Springer Nature remains neutral with regard to jurisdictional rise to several low molecular weight neuropeptides, and claims in published maps and institutional affiliations. 64,65 has been implicated in regulation of energy balance . Electronic supplementary material However, the exact function of the majority of Vgf neu- The online version of this article (https://doi.org/10.1038/s41398-017-0008-3) ropeptides remains elusive. 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Published: Nov 30, 2017

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